Diabetic neuropathy is one of the most important complications that afflict people with diabetes. Because chronic pain caused by diabetic neuropathy is not adequately relieved by existing analgesics, it represents an important unmet clinical need. The cholinergic system in the spinal cord is critically involved in the control of pain transmission. Although distinct M" M3 , and M. subtypes are involved in the regulation of excitatory and inhibitory neurotransmitter release to spinal dorsal horn neurons, little is known about how the function of these mAChR subtypes is altered in diabetic neuropathic pain. The major objectives of this proposal are to study the functional plasticity of spinal muscarinic acetylcholine receptor (mAChR) subtypes in the regulation of nociceptive transmission in painful neuropathy associated with type 1 diabetes. Our preliminary findings suggest that diabetic neuropathy affects primarily the M, and M. mAChR subtypes in the dorsal root ganglion and spinal cord. The specific aims of this project are to determine (1) the functional changes in mAChR subtypes in the spinal dorsal horn and dorsal root ganglion after induction of painful neuropathy in type 1 diabetes and (2) the roles of individual mAChR subtypes in the spinal cord in the control of nociception in diabetic neuropathic pain. Our central hypothesis is that diabetic neuropathy primarily upregulates M, and M. mAChRs on the primary sensory neurons and spinal dorsal horn neurons to inhibit nociceptive transmission. We will use a combination of multidisciplinary approaches including whole-cell patch-clamp recordings of postsynaptic currents in perfused spinal cord slices, real-time RT-PCR, and knockdown of spinal mAChR subtypes with small interfering RNA. These studies will provide substantial novel information about the mechanisms of plasticity in the spinal cholinergic system and mAChR subtypes in diabetic neuropathy. Findings from this project will provide a rationale for the development of new therapies for patients with intractable diabetic neuropathic pain.